Use of Bacillus amyloliquefaciens and / or Bacillus subtilis for enhancing active probiotic compositions or activating inactive probiotic compositions, compositions thus obtained, and related methods

Abstract

The present invention relates to the use of Bacillus amyloliquefaciens and / or Bacillus subtilis for the enhancement of active probiotic compositions or the activation of inactive probiotic compositions for use in the prevention and / or treatment of inflammatory bowel diseases such as Crohn's disease, ulcerative colitis, indeterminate colitis and microscopic colitis, the compositions thus obtained, and methods for the enhancement of active probiotic compositions or the activation of inactive probiotic compositions for use in the prevention and / or treatment of inflammatory bowel diseases such as Crohn's disease, ulcerative colitis, indeterminate colitis and microscopic colitis.

Description

[Technical field]

[0001] The present invention relates to the use of Bacillus amyloliquefaciens and / or Bacillus subtilis for the enhancement of active probiotic compositions or the activation of inactive probiotic compositions for use in the prevention and / or treatment of inflammatory bowel diseases such as Crohn's disease, ulcerative colitis, indeterminate colitis and microscopic colitis, the compositions thus obtained and a method for the enhancement of active probiotic compositions or the activation of inactive probiotic compositions for use in the prevention and / or treatment of inflammatory bowel diseases such as Crohn's disease, ulcerative colitis, indeterminate colitis and microscopic colitis. [Background technology]

[0002] Chronic inflammatory bowel disease, designated by its English acronym IBD or its Italian acronym MICI (Malattie Infiammatorie Croniche dell'Intestino), is a pathological condition that exhibits high morbidity in both adult and pediatric subjects. To date, the available therapies employed to treat such pathologies show only limited efficacy. Although IBD includes various disease patterns, the two most common phenotypes of the disease are Crohn's disease (CD) and ulcerative colitis (UC). Both of these pathologies are chronic relapsing diseases supported by a strong genetic predisposition and immunological etiology, and are usually found in the intestine, although they may also affect other organs, considering that they may be co-expressed in patients with autoimmune diseases such as thyroiditis, sclerosing cholangitis, etc. (de Souza, H.S. and Fiocchi, C., Nat. Rev. Gastroenterol. Hepatol. 13 January 2016(1):13-27).

[0003] Crohn's disease is a transmural intestinal inflammation that may affect any part of the gastrointestinal tract, although the most common location is the terminal ileum, which accounts for approximately 75% of all pathological forms, either alone or in association with a location in the colon (ileocolic disease).

[0004] Ulcerative colitis can also be defined as intermittent inflammation, but without transmural involvement. This condition is typical of the colon, especially the terminal part of the colon (proctitis). In 70% of cases, UC only manifests in the form of ulcerative proctitis (Danese, S. and Fiocchi. C., N. Engl. J. Med. 365:1713-1725, 2011).

[0005] Among the clinical manifestations associated with the presence of colitis, it is possible to identify a subgroup of patients in whom the diagnosis is uncertain, for which the specific term Indeterminate Colitis (IC) has been coined. This condition is characterized by an inflammation limited to the colon, but with histological peculiarities that can be attributed to both Crohn's disease and ulcerative colitis, but which do not belong to either (Brown, CJ et al., Dis. Colon Rectum 48(8):1542-9, 2005).

[0006] The term microscopic colitis ultimately identifies two disorders that are part of chronic inflammatory bowel disease and are characterized by chronic diarrhea: collagenous colitis and lymphocytic colitis. Microscopic colitis affects primarily young women and is often associated with autoimmune diseases such as Sjögren's syndrome, celiac disease and certain types of arthritis. A significant increase in the incidence of microscopic colitis has also been shown in individuals who chronically use certain medications, such as nonsteroidal anti-inflammatory drugs (NSAIDs) and proton pump inhibitors, which significantly alter an individual's microbiota.

[0007] There is now general consensus that IBD originates in genetically predisposed subjects from alterations in the intestinal mucosal barrier that lead to an overload of the local immune system and dysregulation of defense mechanisms, including both innate and adaptive immunity (Baumgart, DC and Sandborn, WJ, Lancet 380:1590-1605, 2011).

[0008] Current treatments are usually based on the use of immunosuppressants such as corticosteroids and azathioprine, and anti-inflammatory drugs such as 5-aminosalicylic acid (Neurath, MF, Nat. Rev. Gastroenterol. Hepatol. 14:269-278, 2017; Tiede, I. et al., J. Clin. Invest. 111:1133-1145, 2003; Lim, WC et al., Cochrane Database Syst. Rev. 7:CD008870, 2016). Recently, biologics with anti-TNF-α activity, such as infliximab, adalimumab, and golimumab, and anti-integrin agents, such as vedolizumab, have been introduced (Neurath, MF, Nat. Rev. Gastroenterol. Hepatol. 14:269-278, 2017; Colombel, JF et al., N. Engl. J. Med. 362:1383-1395, 2010).

[0009] Although the above drugs are highly effective, to date, approximately half of patients are prone to relapse during maintenance therapy, and approximately 30-40% of patients, especially those with CD, do not achieve complete remission or do not maintain remission permanently due to lack of response to treatment or decreasing response over time.

[0010] Furthermore, not infrequently, the phenomenon of intolerance to these drugs and / or the appearance of side effects occurs, leading to the need to interrupt treatment (Mosli, MH et al., Drugs 74:297-311, 2014).

[0011] Furthermore, today, approximately 50% of patients relapse or do not respond to maintenance therapy. Therapeutic approaches have been developed that target the regulation of leukocyte trafficking to the intestine via inhibition of integrins, chemokines and chemokine receptors (Mosli, MH et al., Drugs 74:297-311, 2014).

[0012] One of the studied factors in the pathogenesis of IBD, which has become increasingly important in recent years, is represented by the intestinal microbiota, which constitutes a community of microorganisms including bacteria, fungi, archaea, protozoa and viruses that colonize a specific environment (in this case the intestine) for a certain period of time.

[0013] Alterations in the composition of the intestinal microbiota, termed "dysbiosis", are observed in approximately 75% of patients suffering from CD and may play a pathogenic role in the development of this condition in genetically predisposed subjects (Bellaguarda, E. and Chang, E.B., Curr. Gastroenterol. Rep. 17:15, 2015).

[0014] In this context, the gut microbiota is considered an important therapeutic target in the treatment of IBD.

[0015] It is known that the composition / function of the intestinal microbiota can be modulated by using appropriate probiotics (Chibbar, R. and Dielman, LA, J. Clin. Gastroenterol. 49:S50-S55, 2015).

[0016] Among the numerous probiotic preparations available on the market, the one hereafter referred to as the "De Simone preparation" (U.S. Patent No. 5,716,615), distributed over time under the brand VSL#3 and later under the brand VIVOMIXX and VISBIOME, has been widely studied and recommended for the treatment of chronic pouchitis (Gionchetti, P. et al., Gastroenterology 119:305-309, 2000; Gionchetti, P. et al., Gastroenterology 124(5):1202-9, 2003; Shen, J. et al., Bowel Dis. 20:21-35, 2014). It is a preparation consisting of a mixture of eight different bacterial strains. However, recent in vitro and in vivo studies have shown that similar preparations produced from different raw materials obtained by different culture processes have variable efficacy in reducing inflammation (Cinque, B. et al., J Cell Physiol. 232(12):3530-3539, 2017; Biagioli, M. et al., Front. Pharmacol. 8:505, 2017). Summary of the Invention [Problem to be solved by the invention]

[0017] There is therefore a need to increase scientific research on the intestinal microbiota and to carry out new studies and experiments to create new compositions based on probiotics or to enhance existing ones to obtain increasingly effective compositions in the prevention and / or treatment of inflammatory bowel diseases. [Means for solving the problem]

[0018] Applicant has unexpectedly discovered and experienced that the use of Bacillus amyloliquefaciens and / or Bacillus subtilis allows for the enhancement of already active probiotic compositions as well as the activation of inactive probiotic compositions, resulting in innovative and convenient probiotic compositions that have proven particularly suitable for successful use in the prevention and / or treatment of inflammatory bowel diseases such as Crohn's disease, ulcerative colitis, indeterminate colitis and microscopic colitis.

[0019] The compositions obtained using Bacillus amyloliquefaciens and / or Bacillus subtilis have the following advantages: 1) The use of bacterial species that are less expensive to produce and more stable than traditional probiotics and therefore can be added to both probiotics stored at room temperature and those stored in a refrigerated environment, where the probiotics are formulated in both liquid compositions and granular forms; 2) modification of the functionality of probiotic mixtures with enhanced or activated therapeutic efficacy; 3) Powerful regulation of the intestinal immune system; 4) Strong regulation of enzyme activity.

[0020] Summary of the invention The Applicant has unexpectedly discovered that the use of a probiotic mixture, preferably Bacillus amyloliquefaciens and / or Bacillus subtilis in a total percentage between 2 and 50%, makes it possible to obtain a probiotic blend with a high or enhanced therapeutic effect. The inclusion of Bacillus amyloliquefaciens and / or Bacillus subtilis in a probiotic mixture not only increases the functional activity, and therefore the therapeutic activity, of an already active probiotic mixture, but, surprisingly, makes an inactive probiotic mixture active in reducing intestinal inflammation in the various experimental models investigated. More specifically, the present invention is based on the evidence that the addition of Bacillus amyloliquefaciens and / or Bacillus subtilis to a probiotic mixture, preferably in a total percentage between 2 and 50%, can obtain an increase in anti-inflammatory activity of at least 30% as measured by determining the value of the CDAI (Colitis Disease Activity Index) compared to an equivalent composition without Bacillus amyloliquefaciens and / or Bacillus subtilis, or can obtain an active probiotic mixture if the initial composition was inactive.

[0021] Therefore, an important advantage of compositions containing Bacillus amyloliquefaciens and / or Bacillus subtilis is that the presence of these bacteria in the probiotic activates / increases their function by acting as a probiotic.

[0022] A further important benefit associated with the use of compositions according to this aspect of the invention comprising Bacillus amyloliquefaciens and / or Bacillus subtilis is the increase in regulatory enterocytes (Tregs) present within the colonic lamina propria, which are known for their anti-inflammatory function.

[0023] In a first aspect, the present invention relates to the use of Bacillus amyloliquefaciens and / or Bacillus subtilis for the enhancement of an active probiotic composition or the activation of an inactive probiotic composition in the prevention and / or treatment of inflammatory bowel diseases such as Crohn's disease, ulcerative colitis, indeterminate colitis and microscopic colitis.

[0024] According to the first embodiment of the present invention, the amount of Bacillus amyloliquefaciens and / or Bacillus subtilis added is 2 to 50%.

[0025] According to a second embodiment of the invention, the active probiotic composition to be enhanced is made up of the following eight bacterial strains commercially available under the brands VIVOMIXX and VISBIOME: Lactobacillus paracasei DSM 24733, Lactobacillus plantarum DSM 24730, Lactobacillus acidophilus DSM 24735, Lactobacillus delbrueckii subsp. bulgaricus DSM 24734, Bifidobacterium longum DSM 24736, Bifidobacterium infantis DSM 24737, Bifidobacterium breve DSM 24732, and Streptococcus thermophilus DSM 24731, It is a mixture of.

[0026] According to a third embodiment of the present invention, the active probiotic composition to be enhanced comprises: 20 to 30% by weight of Streptococcus thermophilus, 20-30% by weight of Lactobacillus casei, 30% to 20% by weight of Bifidobacterium breve, and 30% to 20% by weight of Bifidobacterium animalis subsp. lactis, Includes.

[0027] According to a fourth embodiment of the invention, the inactive probiotic composition to be activated is a mixture of the following bacterial strains: Streptococcus thermophilus BT01, Bifidobacterium breve BB02, Bifidobacterium longum (recently reclassified as Bifidobacterium animalis subsp. lactis) BL03, Bifidobacterium infantis (recently reclassified as Bifidobacterium animalis subspecies lactis) BI04, Lactobacillus acidophilus BA05, Lactobacillus plantarum BP06, Lactobacillus paracasei BP07, and Lactobacillus delbrueckii subsp. bulgaricus (recently reclassified as Lactobacillus helveticus) BD08.

[0028] According to a fifth embodiment of the present invention, Bacillus amyloliquefaciens is a known strain characterized by SEQ ID NO:1, which encodes 16S RNA.

[0029] In a second aspect, the present invention relates to an enhanced active probiotic composition or an activated inactive probiotic composition comprising a total of 2-50% by weight of added Bacillus amyloliquefaciens and / or Bacillus subtilis for use in the prevention and / or treatment of inflammatory bowel diseases such as Crohn's disease, ulcerative colitis, indeterminate colitis and microscopic colitis.

[0030] According to a first embodiment of this aspect, an enhanced probiotic composition for use in the prevention and / or treatment of inflammatory bowel diseases such as Crohn's disease, ulcerative colitis, indeterminate colitis and microscopic colitis comprises: 98-50% by weight of the following eight bacterial strains: Lactobacillus paracasei DSM 24733, Lactobacillus plantarum DSM 24730, Lactobacillus acidophilus DSM 24735, Lactobacillus delbrueckii subsp. bulgaricus DSM 24734, Bifidobacterium longum DSM 24736, Bifidobacterium infantis DSM 24737, Bifidobacterium breve DSM 24732, Streptococcus thermophilus DSM 24731, and 2 to 50% by weight of Bacillus amyloliquefaciens and / or Bacillus subtilis; Contains a mixture of.

[0031] According to a second embodiment of this aspect, an activated probiotic composition for use in the prevention and / or treatment of inflammatory bowel diseases such as Crohn's disease, ulcerative colitis, indeterminate colitis and microscopic colitis comprises: 20 to 30% by weight of Streptococcus thermophilus, 10 to 30% by weight of Lactobacillus casei, 10-15% by weight of Bifidobacterium breve, 10-15% by weight of Bifidobacterium animalis subsp. lactis, and 50-10% by weight of Bacillus amyloliquefaciens and / or Bacillus subtilis; Includes.

[0032] According to a third embodiment of this aspect, the enhanced probiotic composition for use in the prevention and / or treatment of inflammatory bowel diseases such as Crohn's disease, ulcerative colitis, indeterminate colitis and microscopic colitis comprises: 98-50% by weight of the following eight bacterial strains: Streptococcus thermophilus BT01, Bifidobacterium breve BB02, Bifidobacterium longum (recently reclassified as Bifidobacterium animalis subsp. lactis) BL03, Bifidobacterium infantis (recently reclassified as Bifidobacterium animalis subspecies lactis) BI04, Lactobacillus acidophilus BA05, Lactobacillus plantarum BP06, Lactobacillus paracasei BP07, Lactobacillus delbrueckii subsp. bulgaricus (recently reclassified as Lactobacillus helveticus) BD08, and 2 to 50% by weight of Bacillus amyloliquefaciens and / or Bacillus subtilis; Contains a mixture of.

[0033] According to a fourth embodiment of this aspect, the Bacillus amyloliquefaciens is a known strain characterized by SEQ ID NO:1, which encodes the 16S RNA.

[0034] In a third aspect, the present invention provides a method for the prevention and / or treatment of inflammatory bowel diseases, such as Crohn's disease, ulcerative colitis, indeterminate colitis and microscopic colitis, comprising: 20 to 30% by weight of Streptococcus thermophilus, 20-30% by weight of Lactobacillus casei, 30% to 20% by weight of Bifidobacterium breve, and 30% to 20% by weight of Bifidobacterium animalis subsp. lactis, The present invention relates to a novel probiotic composition comprising:

[0035] In a fourth aspect, the present invention relates to a method for the enhancement of an active probiotic composition or for the activation of an inactive probiotic composition in the prevention and / or treatment of inflammatory bowel diseases such as Crohn's disease, ulcerative colitis, indeterminate colitis and microscopic colitis, comprising the addition of Bacillus amyloliquefaciens and / or Bacillus subtilis to such a probiotic composition.

[0036] According to a first embodiment of this aspect, the weight amount of Bacillus amyloliquefaciens and / or Bacillus subtilis added is between 2 and 50%.

[0037] According to a second embodiment of this aspect, the enhanced active probiotic composition contains the following eight bacterial strains: Lactobacillus paracasei DSM 24733, Lactobacillus plantarum DSM 24730, Lactobacillus acidophilus DSM 24735, Lactobacillus delbrueckii subsp. bulgaricus DSM 24734, Bifidobacterium longum DSM 24736, Bifidobacterium infantis DSM 24737, Bifidobacterium breve DSM 24732, and Streptococcus thermophilus DSM 24731, It is a mixture of.

[0038] According to a third embodiment of this aspect, the active probiotic composition to be enhanced comprises: 20 to 30% by weight of Streptococcus thermophilus, 20-30% by weight of Lactobacillus casei, 30% to 20% by weight of Bifidobacterium breve, and 30% to 20% by weight of Bifidobacterium animalis subsp. lactis, Includes.

[0039] According to a fourth embodiment of this aspect, the inactive probiotic composition to be activated contains the following seven bacterial strains: Streptococcus thermophilus BT01, Bifidobacterium breve BB02, Bifidobacterium longum (recently reclassified as Bifidobacterium animalis subsp. lactis) BL03, Bifidobacterium infantis (recently reclassified as Bifidobacterium animalis subspecies lactis) BI04, Lactobacillus acidophilus BA05, Lactobacillus plantarum BP06, Lactobacillus paracasei BP07, and Lactobacillus delbrueckii subsp. bulgaricus (recently reclassified as Lactobacillus helveticus) BD08, It is a mixture of.

[0040] According to a fifth embodiment of this aspect, the Bacillus amyloliquefaciens is a known strain characterized by SEQ ID NO:1, which encodes the 16S RNA. [Brief description of the drawings]

[0041] [Fig. 1A-1F]Figures 1A-1F show the enhanced protective effect that accompanies the administration of Bacillus amyloliquefaciens species in addition to a bacterial composition already active in this effect. In particular, Figure 1A shows the trend in body weight, Figure 1B shows the daily measurements of the Colitis Disease Activity Index (CDAI) that takes into account weight loss, stool consistency and the presence of blood in the stool, Figure 1C shows the colon length, Figure 1D shows the ratio of colon weight to colon length, and Figures 1E and 1F show the percentage of immune cells present in the lamina propria of the colon (Figure 1E: CD3+IL-10+ T lymphocytes within CD3+ T lymphocytes; Figure 1F: M2 CD11b+Gr1-IL-10+ macrophages within CD11+Gr1- macrophages). Results are expressed as the mean ± SEM of 7-10 mice per experimental group. *p <0.05. [Figures 2A-2E] Figures 2A-2E show the enhanced protective effect that accompanies the administration of Bacillus amyloliquefaciens species in addition to further bacterial compositions already active in this effect. In particular, Figure 2A shows the trend in body weight, Figure 2B shows the daily measurements of CDAI, which considers weight loss, stool consistency and the presence of blood in the stool, Figure 2C shows the ratio between colon weight and colon length, and Figures 2D and 2E show the frequency of Treg CD4+FoxP3+ immune cells present in the lamina propria of the colon (Figure 2D: within CD4+ T lymphocytes; Figure 2E: within all cells of the colonic lamina propria). Results are expressed as mean ± SEM of 6-8 mice per experimental group. *p <0.05. [Figure 3A-3B] Figures 3A-3B show the protective effect associated with the administration of Bacillus amyloliquefaciens species compared to a bacterial composition that does not show this effect. In particular, Figure 3A shows the trend in body weight and Figure 3B shows the daily measurements of CDAI, which considers weight loss, stool consistency and the presence of blood in the stool. Results are expressed as the mean ± SEM of 6-8 mice per experimental group. *p <0.05. [Figure 4A-4B]Figures 4A-4B show the effect of Bacillus amyloliquefaciens alone as well as in combination with some of the compositions of the present invention. In particular, Figure 4A shows the trend in body weight and Figure 4B shows daily measurements of CDAI, which considers weight loss, stool consistency and the presence of blood in the stool. Results are expressed as the mean ± SEM of 6-8 mice per experimental group. *p < 0.05. [Figure 5A-5B] Figures 5A-5B show the substantial equivalence of the effects of Bacillus amyloliquefaciens and Bacillus subtilis in the tests performed. In particular, Figure 5A shows the trend in body weight and Figure 5B shows the daily measurements of CDAI, which considers weight loss, stool consistency and the presence of blood in the stool. Results are expressed as the mean ± SEM of 6-8 mice per experimental group. *p <0.05. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] Detailed Description of the Invention Experimental part material and method The strain of Bacillus amyloliquefaciens used in the experiments is a known strain characterized by SEQ ID NO:1, which encodes the 16S RNA.

[0043] A murine model of colitis induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS) was used to test the anti-inflammatory activity of the tested compositions. In this model, colonic inflammation is induced by intrarectal administration of TNBS in a 50% ethanol solution. Ethanol is essential to provide access to intestinal epithelial cells and impair barrier function, allowing TNBS to penetrate the intestinal wall.

[0044] The model itself is suitable for investigating the role of T cells in intestinal inflammation, since it is associated with an increased presence of highly activated T cells, which play an important pathogenic role in TNBS-induced colitis (Elson, CO et al., J. Immunol. 157:2174-2185, 1996; Dohi, T. et al., J. Exp. Med. 189:1169-1180, 1999). Nevertheless, immune mechanisms are also involved in the development of TNBS colitis (Fiorucci, S. et al., Immunity 17:769-780, 2002).

[0045] Histologically, disease induction is followed by transmural inflammation with infiltration of macrophages, neutrophils and lymphocytes, as well as colonic hypertrophy (Kiesler, P. et al., Cell. Mol. Gastroenterol. Hepatol. 1:154-170, 2015). Because several immunological and histopathological features resemble those associated with Crohn's disease, TNBS-induced colitis has been widely used to study various aspects that may be related to this disease (Kiesler, P. et al., Cell. Mol. Gastroenterol. Hepatol. 1:154-170, 2015; Rieder, F. et al., Gut 62:1072-1084, 2013).

[0046] Eight-week-old C57BL6 male mice were purchased from the Jackson Laboratory and were maintained at the Preclinical Research Center of the University of Perugia.

[0047] Mice were maintained at a controlled temperature of 22 °C with a 12 / 12 h light / dark period and were allowed to acclimate under these conditions for 7 days before use in experimental activities. The study was carried out in accordance with Italian legislation and the experimental protocol was approved by the Ethics Committee of the University of Perugia and the Ministry of Health (permit number 1126 / 20-PR).

[0048] For induction of colitis, mice were fasted for 12 hours (day -1). On the following day (day 0), mice were anesthetized and a catheter was inserted into the colon up to 4 cm from the anus. To induce colitis, 1 mg of TNBS dissolved in 50% ethanol was administered through the catheter with a 1 ml syringe (injection volume: 100 μl).

[0049] Control animals (untreated, NT) received only 50% ethanol solution. Mice were then monitored daily to assess the course of the disease by measuring body weight and the CDAI (Colitis Disease Activity Index), which includes weight loss, stool consistency and the presence of blood in the stool (values ​​for each parameter range from 0 to 4).

[0050] At the end of the experiment, animals were sacrificed and colons were harvested for analysis of gross characteristics and for extraction of lamina propria cells with the Lamina Propria Dissociation Kit Mouse (Miltenyi Biotec) for analysis by cytofluorometry.

[0051] The probiotic composition was 50×10 9 Probiotic concentrations of cfu / kg body weight were administered daily (from day 0 to day 4) via oral probe. EXAMPLES

[0052] In this example, the following eight recent strains, commercially available under the VIVOMIXX brand, were used: Lactobacillus paracasei DSM 24733, Lactobacillus plantarum DSM 24730, Lactobacillus acidophilus DSM 24735, Lactobacillus delbrueckii subsp. bulgaricus DSM 24734, Bifidobacterium longum DSM 24736, Bifidobacterium infantis DSM 24737, Bifidobacterium breve DSM 24732, and Streptococcus thermophilus DSM 24731, Compositions active in the prevention and / or treatment of inflammatory diseases, comprising:

[0053] Figure 1 shows the results of the test carried out according to the above method, where NT = untreated and TNBS = treated with 2,4,6-trinitrobenzenesulfonic acid only.

[0054] The experimental data results clearly show that in the TNBS-induced colitis model, the addition of Bacillus amyloliquefaciens improves the efficacy of VIVOMIXX in preventing the attenuation of weight loss, inducing an improvement in inflammation measured as CDAI, and in attenuating TNBS-induced colonic morphological changes, such as the ratio of colonic weight to colonic length. An increase in regulatory intestinal cells (Treg) within the colonic lamina propria is also evident. EXAMPLES

[0055] In this example, novel compositions were tested that were found to be effective in the prevention and / or treatment of inflammatory diseases.

[0056] The novel composition comprises: 30% by weight of Streptococcus thermophilus, 30% by weight of Lactobacillus casei, 20% by weight of Bifidobacterium breve, and 20% by weight of Bifidobacterium animalis subsp. lactis, Includes.

[0057] 2A-2E show the results of tests performed according to the methods described above, where NT = untreated, TNBS = treated with 2,4,6-trinitrobenzenesulfonic acid only.

[0058] The results of the experimental data using the above composition clearly show that the composition exhibits excellent efficacy in preventing the reduction of weight loss, inducing an improvement in inflammation measured as CDAI, and attenuating TNBS-induced colonic morphological changes, such as the ratio of colonic weight to colonic length, in the TNBS-induced colitis model. An increase in regulatory intestinal cells (Treg) in the colonic lamina propria is also evident.

[0059] To determine the effect of adding Bacillus amyloliquefaciens to this composition, the composition was modified as follows: 30% by weight of Streptococcus thermophilus, 30% by weight of Lactobacillus casei, 15% by weight of Bifidobacterium breve, 15% by weight of Bifidobacterium animalis subsp. lactis, and 10% by weight of Bacillus amyloliquefaciens.

[0060] The results of the experimental data summarized in Figures 2A-2E clearly show that in the TNBS-induced colitis model, the addition of B. amyloliquefaciens improves the efficacy of the novel composition in preventing the reduction of body weight loss, inducing an improvement in inflammation measured as CDAI, and in attenuating the morphological changes in the colon induced by TNBS, such as the ratio of colon weight to colon length. An increase in regulatory intestinal cells (Treg) within the colonic lamina propria is also evident. EXAMPLES

[0061] In this example, the following bacterial strains were used: Streptococcus thermophilus BT01, Bifidobacterium breve BB02, Bifidobacterium longum (recently reclassified as Bifidobacterium animalis subspecies lactis) BL03, Bifidobacterium infantis (recently reclassified as Bifidobacterium animalis subspecies lactis) BI04, Lactobacillus acidophilus BA05, Lactobacillus plantarum BP06, Lactobacillus paracasei BP07, and Lactobacillus delbrueckii subsp. bulgaricus (recently reclassified as Lactobacillus helveticus) BD08, A commercially available composition containing was tested.

[0062] As can be seen from the results shown in FIG. 3, this composition was inactive in the tests performed, likely due to differences in the culture procedures of the strains.

[0063] Nevertheless, the addition of B. amyloliquefaciens created a composition that was effective in preventing weight loss as well as in inducing an improvement in inflammation as measured by CDAI.

[0064] conclusion Figures 4A-4B reveal an enhanced protective effect associated with administering the bacterial species Bacillus amyloliquefaciens in addition to a bacterial composition already active with respect to this effect, or alternatively, the establishment of protective activity with administration of the same species in addition to a bacterial composition that does not inherently exhibit this characteristic.

[0065] Furthermore, Figures 5A-5B show the substantial equivalence of the effects associated with the administration of the bacterial species Bacillus amyloliquefaciens and Bacillus subtilis, which is explained by the high degree of genetic similarity between the two groups of bacteria.

[0066] In fact, Bacillus amyloliquefaciens and Bacillus subtilis constitute two sister species (species derived from a shared ancestor) that belong to a larger group of bacterial species characterized by a high genetic correlation called the Bacillus subtilis group (Fritze, D., "Taxonomy of the genus Bacillus and related genera: the aerobic endospore-forming bacteria", Phytopathology 94:1245-1248, 2004). The high genetic similarity between the species B. amyloliquefaciens and B. subtilis, the marker of choice for the taxonomic identification of bacteria (Woese, C.R., "Bacterial evolution", Microbiol. Rev. 51:221-271, 1987), is clearly demonstrated by the difficulty and / or impossibility of efficiently distinguishing these two bacterial groups by comparing the sequences coding for 16S rRNA (Wang et al., "Bacillus velezensis is a later heterotypic synonym of Bacillus amyloliquefaciens", Int. J. Syst. Evol. Microbiol. 58:671-675, 2008). In recent years, the development of a new generation of sequencing technologies has made it possible to access a large amount of data on the sequences of the whole genomes of many bacterial species and to develop investigative methods that allow a more reliable and accurate assessment of the genetic similarity between microbial groups. In this context, conserved genome-wide extended multiple loci based genomic typing (core-MLST) is a powerful tool to determine correlations between different taxa by simultaneously comparing hundreds or even thousands of genetic markers shared between different taxa. Application of this technique to specific bacterial strains belonging to the species B. amyloliquefaciens and B. subtilis further highlights the great genetic similarity between these taxa.The reconstruction of the phylogenetic relationships between the genomes belonging to the two species (see dendrogram below), based on the alignment of the 1664-gene cluster determined using a sequence similarity threshold of ≥80%, places B. amyloliquefaciens and B. subtilis strains phylogenetically closer to each other than to strains within their own taxonomic groups (groups A, B, C, D).

[0067] [Table 1]

[0068] Dendrogram of the reconstruction of the phylogenetic relationships between B. amyloliquefaciens and B. subtilis strains based on a comparison of 1664 genetic markers shared by the two species. The genealogical distance, defined as the number of derived trait changes between the two taxa, is reported for each arm of the phylogenetic tree.

[0069] The high genetic correlation, together with the large number of genes shared between the two bacterial species, allows to assume that they might also be equivalent from a functional point of view.

[0070] material and method Genome typing based on multiple loci extended across the core genome was performed on genomes related to B. amyloliquefaciens and B. subtilis species available in public databases (ftp: / / ftp.ncbi.nlm.nih.gov / genbank). Five complete genome sequences were selected for each bacterial species, including those related to the species type strains B. amyloliquefaciens DSM7 and B. subtilis ATCC13952. To avoid the introduction of errors due to different genome annotations, all sequences were de novo annotated by the Prokka v1.12 pipeline (Seeman T, "Prokka: rapid prokaryotic genome annotation", Bioinformatics 15 Jul. 2014, 30(14):2068-9), which enforces compliance with the Genbank / ENA / DDJB standard. Genomes were imported into the BPGA bioinformatics program (Chaudhari et al., "BPGA-an ultra-fast pan-genome analysis pipeline" Sci. Rep. 6:24373, 2016) and homologous gene groups were determined using a threshold of 80% or greater similarity between protein sequences encoded by gene sequences. Homologous protein groups were aligned separately using MUSCLE v 3.8.31 (Edgar, R.C., "MUSCLE: multiple sequence alignment with high accuracy and high throughput", Nucleic Acids Res. 32:1792-7, 2004) and subsequently joined to form concatemers. Phylogenetic reconstructions were performed using approximate maximum likelihood methods using the Fasttree2 program (Price et al., "FastTree 2-Approximately maximum-likelihood trees for large alignments", PLoS One 5:e9490, 2010). Figures 5A-5B also show that the effectiveness of Bacillus amyloliquefaciens and Bacillus subtilis was substantially equivalent in the tests performed.

Claims

1. 2. Use of Bacillus amyloliquefaciens and / or Bacillus subtilis for the manufacture of a probiotic composition for the prevention and / or treatment of inflammatory bowel diseases, such as Crohn's disease, ulcerative colitis, indeterminate colitis and microscopic colitis, comprising The probiotic composition contains the following eight bacterial strains: Lactobacillus paracasei DSM 24733, Lactobacillus plantarum DSM 24730, Lactobacillus acidophilus DSM 24735, Lactobacillus delbrueckii subsp. bulgaricus DSM 24734, Bifidobacterium longum DSM 24736, Bifidobacterium infantis DSM 24737, Bifidobacterium breve DSM 24732, Streptococcus thermophilus DSM 24731, a mixture of and 2 to 50% by weight of Bacillus amyloliquefaciens and / or Bacillus subtilis; The use, which is a composition comprising:

2. 2. Use of Bacillus amyloliquefaciens and / or Bacillus subtilis for the manufacture of a probiotic composition for the prevention and / or treatment of inflammatory bowel diseases, such as Crohn's disease, ulcerative colitis, indeterminate colitis and microscopic colitis, comprising 1. A probiotic composition comprising: 20-30% by weight of Streptococcus thermophilus, 20-30% by weight of Lactobacillus casei; 30 to 20% by weight of Bifidobacterium breve, 30-20% by weight of Bifidobacterium animalis subsp. lactis, and 2 to 50% by weight of Bacillus amyloliquefaciens and / or Bacillus subtilis; The use, which is a composition comprising:

3. 2. Use of Bacillus amyloliquefaciens and / or Bacillus subtilis for the manufacture of a probiotic composition for the prevention and / or treatment of inflammatory bowel diseases, such as Crohn's disease, ulcerative colitis, indeterminate colitis and microscopic colitis, comprising The probiotic composition comprises the following bacterial strains: Streptococcus thermophilus BT01, Bifidobacterium breve BB02, Bifidobacterium longum (recently reclassified as Bifidobacterium animalis subsp. lactis) BL03, Bifidobacterium infantis (recently reclassified as Bifidobacterium animalis subspecies lactis) BI04, Lactobacillus acidophilus BA05, Lactobacillus plantarum BP06, Lactobacillus paracasei BP07, A mixture of Lactobacillus delbrueckii subsp. bulgaricus (recently reclassified as Lactobacillus helveticus) BD08, and 2 to 50% by weight of Bacillus amyloliquefaciens and / or Bacillus subtilis; The use, which is a composition comprising:

4. The use according to any one of claims 1 to 3, wherein a known strain of Bacillus amyloliquefaciens is used, characterized by SEQ ID NO: 1 which codes for the 16S RNA.

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